Technical Abstract:
Research on tree nut crops has demonstrated that aflatoxin (AF) biosynthesis appears to be inhibited by gallic acid (GA). It is hypothesized that the release of GA from hyrolyzable tannins present in the plant seed coat or hull tissue by fungal tannase enzyme is responsible for the observed inhibition of AF production. We have shown that inhibition of AF production appears to be at the level of transcription of AF biosynthetic genes since GA reduced expression of the aflatoxin biosynthetic genes nor1, ver1, and omtA genes, although aflR (positive regulator of AF gene expression) expression was only slightly reduced. In order to test if GA is the major inhibitory compound in the seed coat/hull tissues, we cloned the tannase gene from A. flavus. A tannase gene disruption construct was engineered and transformed into an A. flavus 70 niaD mutant and one isolate, 13B, exhibited loss of tannase activity. We examined if inhibition of toxin production is less in the tannase knockout mutant due to its inability to release GA from tannic acid (TA).
Both the wild-type A. flavus 70 and the tannase knockout mutant 13B were grown for 3 days on minimal salts/1% sucrose plates supplemented with 0, 0.05, 0.125, or 0.25% GA or TA. Essentially no difference in radial growth was observed when comparing the wild-type to 13B on media supplemented with GA. No significant differences were observed between the two fungi for growth on TA either. However, significant differences were observed when comparing fungal growth on GA vs. TA. Radial growth of both isolates was the same at 0 and 0.05% GA or TA but reduced by 13.6% and 37.5% on 0.125 and 0.25% GA respectively, compared to TA. Slightly greater inhibition of AF production was observed in 13B compared to wild-type at higher GA concentrations. Data will also be presented on differences in response to GA or TA by L- and S-strains of A. flavus. Growth, aflatoxin production and transcription of aflatoxin pathway genes of A. flavus 13 (L-strain), 70 (S-strain), and the A. flavus 70-13B tannase knockout mutant in the presence of TA or GA will be reported.